Novel Biomarkers in Pediatric MS Using Proteomics

To determine if the pattern of protein-expression profiles in pediatric multiple sclerosis (MS) differs from age matched healthy controls (HC) and other medical disease controls(OMD).

To identify a subset of MS specific proteins in plasma not seen in controls HC which could serve as predictive biomarkers of the disease.

Background

Proteomics has great potential for identifying early biomarkers of disease states and could offer a sensitive diagnostic tool for MS. A particularly attractive feature is that proteomics can be applied to the plasma of affected patients providing a relatively non-invasive and highly feasible approach. The advent of gel-image analysis software (e.g. PDQuest) has revolutionized two dimensional electrophoresis (2DE) gel based technology. Once novel or statistically different levels of protein expression are found they can be subjected to mass spectrometry for protein identification, which might lead to improved diagnosis and prognosis. Previously we had identified 12 proteins from the serum which differed in expression in MS relative to HC. However, we have further improved the sensitivity of our proteomics approach for the detection of low abundance proteins and we have expanded our studies to include a medical control group in addition to HC. Herein we report the results of our proteomic approach designed to detect low abundance proteins and describe MS specific findings relative to other medical controls.

Methods

Subjects:
Age matched pediatric MS (n=6);HC (n=4), and OMD controls (n=4) including:
one somatoform disorder, two mitochondrial disorders, and one with a cardiovascular disorder.

Proteomics:
Serum from subjects is depleted of 20 high abundance proteins so that low abundance protein proteins can be detected.
Long range 2D gels were used to separate proteins by pI and MW.
Normalization (using log transformation) of the data prior to statistical analysis which optimizes the reliability of the comparison of protein expression levels from protein spots among MS, HC and OMD control groups.
Application of the LTQ XL ion trap mass spectrometry for protein identification. The LTQ XL ion trap is a powerful tool for protein identification and characterization due to its capabilities of high resolution, rapid scan, and its excellent mass accuracy in a robust manner even within HPLC time scales.

Results

Shown in figure 1 is the master gel of proteins

As shown in figure 2, different sequences of haptoglobin isoforms 1 and 2 were detected in the MS but not in the control subjects.
There were increases in expression levels of several other proteins which included the precursor to Vitamin D binding protein, transthyretin.

Our method also allowed detection of important low abundance proteins which had an increased expression level in pediatric MS (including vitronectin, glutathione peroxidases) and decreased level (lumican) relative to OMD and HC.